Iron is an essential micronutrient for nearly all bacteria, and sequestration of iron by the mammalian host poses significant problems for an invading bacterial pathogen. Despite the central role of iron acquisition in bacterial growth, virtually nothing is known about how F. tularensis acquires this important cation and, to date, there have been no descriptions of F. tularensis gene products that are involved in this process. In other well-studied prokaryotic parasites, growth under iron-restricted conditions results in the expression of a myriad of bacterial gene products, some of which enhance the virulence potential of their respective pathogens. The Specific Aims of this proposal are designed to address this gap in our knowledge about this pathogen that represents a serious threat as a bioterrorism weapon. Identification of gene products involved in iron acquisition by the virulent F. tularensis Schu4 strain will be accomplished using a number of complementary approaches including both genomic and proteomic methodologies. The sources of iron that can be utilized by F. tularensis Schu4 and its possible production of a siderophore will be investigated in Specific Aim 1. We will use two-dimensional gel electrophoresis, DNA microarray analysis, and a GFP-based promoter trap in Specific Aim 2 to identify those F. tularensis Schu4 proteins whose expression is induced or up-regulated as a result of iron limitation. We will construct isogenic F. tularensis Schu4 mutants that lack the ability to express these proteins and determine whether these mutants have an altered ability to acquire iron, to survive and grow in human macrophages, or to cause disease in a murine model of pulmonary tularemia in Specific Aim 3. We will also determine whether iron-regulated outer membrane proteins (identified in Specific Aim 1) have the ability to induce protective immunity against aerosol challenge with F. tularensis Schu 4 in Specific Aim 4. These results will determine whether one or more of these iron-regulated proteins might have potential for vaccine development.